We have identified novel small compound inhibitors of flavivims replication. Our most potent compound has a 50% effective concentration (EC50) of 0.7 micromolar and a 50% cytotoxic concentration (CC50) of >70 micromolar, yielding a therapeutic (selectivity) index of >100. We have demonstrated that the compound protects animals from the lethality of WNV infection and reduces viral RNA levels over a hundred-fold in infected cells;and that analogues inhibit replication of related flaviviruses such as the select agent Japanese encephalitis virus (JEV) and the category A biodefense pathogen Dengue virus (DENV). The development of our small molecule antivirals will provide a new approach to the treatment of flavivirus infections, and will complement vaccine approaches. Moreover, because very few putative flavivirus inhibitors have been described, our work is essential to establish a foundation for understanding structure-activity relationships and how these compounds influence host cell functions and innate immunity. To achieve these goals, we propose to analyze the antiviral activities and potential toxicities of small compound analogues related to our inhibitors;to characterize their antiviral mechanisms;and to examine their effects on host cells. Our specific aims are as follows: 1 .Analysis of small molecule anti-flavivirus activities: Small libraries of flavivirus inhibitors and related analogues will be prepared, and compound cellular toxicities and antiviral effects against flavivirus strains will be quantitated. These studies will determine chemical and strain requirements for virus inhibition. 2. Characterization of the mechanisms of compound inhibition: The mechanisms of viral inhibition will be characterized. Examination of compound effects on wild type infections and replicon expression will help delineate inhibitor activities, and assays on viral protein activities will help define inhibition mechanisms. Analysis of putative drug-resistant mutants will help assess whether resistance is acquired at a fitness cost to the virus, and will identify genes targeted by inhibitors. Our results will establish a mode of antiviral action, and ways to optimize inhibitor activities. 3. Examination of inhibitor effects on host cells: Although our most effective flavivirus inhibitors are non-toxic, their specific effects on host cells are almost completely unknown. To fill this gap, we will monitor compound effects on infected and uninfected cells. These results will provide crucial data as to how our novel class of flavivirus inhibitors influence cellular expression profiles and innate immune response mechanisms